The research proposed in this competing grant renewal focuses on dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP), two non-collagenous extracellular matrix (ECM) proteins that belong to the SIBLING family. The importance of DMP1 and DSPP in biomineralization has been demonstrated by studies showing that humans or mice with altered Dmp1 or Dspp gene display defects in the mineralization of bone and/or dentin. Although solid data have demonstrated the association between these two proteins and the formation of healthy bone and/or dentin, the specific mechanistic pathways by which DMP1 and DSPP participate in osteogenesis and dentinogenesis remain ill-defined. Studies proposed in this application are aimed to test how posttranslational proteolytic processing of DMP1 and DSPP affects the mineralization of bone and/or dentin. The following data from our independent and collaborative studies have established that DMP1 and DSPP undergo proteolytic processing and that posttranslational processing may be essential for the mineralization of bone and/or dentin. Our findings include: 1) DMP1 and DSPP are present in the ECM of bone and dentin as NH2-terminal and COOH-terminal fragments. 2) Proteolytic processing of DMP1 and DSPP involves cleavage of peptide bonds at the NH2-termini of aspartyl residues (i.e., at X-Asp bonds). 3) The highly glycosylated NH2-terminal fragments of DMP1 and DSPP have biochemical features distinct from those of their respective COOH-terminal fragments. 4) The highly phosphorylated COOH-terminal fragments of DMP1 and DSPP promote mineralization in vitro. 5) Substitution of an aspartyl residue at a cleavage site blocks the proteolytic processing of DMP1 in transfected eukaryotic cells. 6) Re-expression of the COOH-terminal fragment of DMP1 is capable of rescuing the mineralization defects in Dmp1-null mice. 7) The NH2-terminal fragment and COOH-terminal fragment of DMP1 show clear difference in tissue localization. We hypothesize that proteolytic processing of DMP1 and DSPP by cleavage of selected X-Asp bonds is an activation step that converts precursors from inactive forms to fragments that are fully active at the correct time and site to control the mineralization process. These processing events play crucial roles in the mechanisms that control the conversion of osteoid to bone and predentin to dentin. Hence, failure to process these two proteins will lead to abnormal bone and/or dentin formation. To test the above hypothesis, we propose a series of studies to determine if failure to process these two proteins due to substitution of Asp residues at key cleavage sites will result in the loss of DMP1 or DSPP functions in vivo, and to characterize the roles of the COOH-terminal fragment and NH2-termnial fragment of DSPP. The proposed studies are expected to unfold greater understanding of the process controlling osteogenesis and dentinogenesis. PUBLIC HEALTH RELEVANCE: Fundamental information about how DMP1 and DSPP affect the formation of bone and dentin is essential to enhanced understanding concerning the pathogeneses of bony and dental defects associated with systemic diseases such as osteomalacia, osteoporosis and dentinogenesis imperfecta. Results of the studies proposed in this application will unfold greater understanding of the mechanisms controlling the mineralization of bone and dentin, thus proving information needed for delineating the pathogeneses underlying these bony and dental defects and for establishing scientifically based treatment modalities for such skeletal diseases.